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ORIGINAL RESEARCHpublished: 24 January 2019
doi: 10.3389/fchem.2018.00655
Frontiers in Chemistry | www.frontiersin.org 1 January 2019 | Volume 6 | Article 655
Edited by:
Erik Reimhult,
University of Natural Resources and
Life Sciences Vienna, Austria
Reviewed by:
Allen Liu,
University of Michigan, United States
Jae-Byum Chang,
Korea Advanced Institute of Science &
Technology (KAIST), South Korea
*Correspondence:
Eva Sevcsik
Specialty section:
This article was submitted to
Nanoscience,
a section of the journal
Frontiers in Chemistry
Received: 31 October 2018
Accepted: 17 December 2018
Published: 24 January 2019
Citation:
Lindner M, Tresztenyak A, Fülöp G,
Jahr W, Prinz A, Prinz I, Danzl JG,
Schütz GJ and Sevcsik E (2019) A
Fast and Simple Contact Printing
Approach to Generate 2D Protein
Nanopatterns. Front. Chem. 6:655.
doi: 10.3389/fchem.2018.00655
A Fast and Simple Contact PrintingApproach to Generate 2D ProteinNanopatternsMarco Lindner 1,2, Aliz Tresztenyak 2, Gergö Fülöp 1, Wiebke Jahr 3, Adrian Prinz 2,
Iris Prinz 2, Johann G. Danzl 3, Gerhard J. Schütz 1 and Eva Sevcsik 1*
1 Institute of Applied Physics, TU Wien, Vienna, Austria, 2 Stratec Consumables GmbH, Anif, Austria, 3 Institute of Science and
Technology Austria, Klosterneuburg, Austria
Protein micropatterning has become an important tool for many biomedical applications
as well as in academic research. Current techniques that allow to reduce the feature
size of patterns below 1µm are, however, often costly and require sophisticated
equipment. We present here a straightforward and convenient method to generate
highly condensed nanopatterns of proteins without the need for clean room facilities
or expensive equipment. Our approach is based on nanocontact printing and allows for
the fabrication of protein patterns with feature sizes of 80 nm and periodicities down to
140 nm. This was made possible by the use of the material X-poly(dimethylsiloxane) (X-
PDMS) in a two-layer stamp layout for protein printing. In a proof of principle, different
proteins at various scales were printed and the pattern quality was evaluated by atomic
force microscopy (AFM) and super-resolution fluorescence microscopy.
Keywords: contact printing, protein patterning, nanopatterns, nanofabrication, super-resolution fluorescence
microscopy, STED microscopy
INTRODUCTION
In recent years, surfaces featuring micropatterns of biomolecules, particularly proteins, haveseen a surge of interest. They have found multiple applications in biomedical research such asmicroarrays (Macbeath and Schreiber, 2000; Allison et al., 2006; Wingren and Borrebaeck, 2007),proteomics (Haab, 2005; Wingren and Borrebaeck, 2008), and biomimetic sensors (Mujahid et al.,2013). Furthermore, cell-scale microstructured biointerfaces have been used to manipulate cellshape and organization to study endocytosis (Tan et al., 2015), cell polarization and proliferation(Chen et al., 1997; Théry, 2010), host-pathogen interactions (March et al., 2015) as well asstem cell differentiation (Sykova and Forostyak, 2013; Castaño et al., 2014). On a smaller scale,micropatterned surfaces have been applied to influence the protein distributionwithin living cells toaddress several cell biological questions such as plasmamembrane organization (Sevcsik et al., 2015;Fülöp et al., 2018), dynamics of cytokine receptor signaling (Löchte et al., 2014), T cell signaling(Mossman et al., 2005), protein-protein interactions (Lanzerstorfer et al., 2014; Dirscherl et al.,2018), phagocytosis (Freeman et al., 2016), and cell adhesion (Schvartzman et al., 2011; Coyer et al.,2012). Different techniques have been developed for fabricating patterned surfaces which cater tothe demands of the respective applications.
One family of techniques is based on indirect deposition of proteins; the most prominent ofthese are photolithography (Christman et al., 2006; Lenci et al., 2011) and laser microablation(Nicolau et al., 2010), where the minimum feature sizes are set by the diffraction limit of light.
Lindner et al. 2D Protein Nanopatterns
This restriction can be overcome by colloidal lithography(Kristensen et al., 2013), di-block copolymer micellenanolithography (Thelen et al., 2007; Lohmüller et al.,2011) or di-block copolymer self-assembly (Hortigüelaet al., 2017). A different approach of indirect depositionemployed electropolymerization to functionalize gold micro-and nanoelectrodes with proteins (Della Pia et al., 2014).Methods based on direct deposition of proteins include masklessprojection lithography (Waldbaur et al., 2012), microfluidicpatterning (Delamarche et al., 1998; Alom Ruiz and Chen, 2007),and contact-based printing. Here, one of the most convenient,cost-efficient, and widely used methods has been microcontactprinting (µCP) with poly(dimethylsiloxane) (PDMS) stamps(Wilbur et al., 1994; Bernard et al., 2000). PDMS has severalproperties that made it a perfect choice for a stamp material: it iselastomeric and chemically inert, it molds with high fidelity andis, due to its low surface energy, easily removed from the moldafter curing as well as from the substrate after printing (AlomRuiz and Chen, 2007).
While several convenient methods exist today to createmicrostructured surfaces, the fabrication of patterns with afeature size below 1µm is still challenging and often requirestrade-offs between speed, biocompatibility, cost, versatility, andexperimental complexity. Easy availability of high-performancenano-biointerfaces would open up completely new vistas forproteomics and cell research: in case of protein biochips, thetotal chip surface and hence the required amount of samplematerial for analysis could be massively reduced; using nano-biointerfaces as substrates for living cells allows for investigatingthe influence of protein organization at the nanoscale onsignaling (Deeg et al., 2013; Shaw et al., 2014; Cai et al., 2018),adhesion (Arnold et al., 2004; Cavalcanti-Adam et al., 2007),or cell differentiation (Wang et al., 2015). Additionally, nm-sized structures fit capabilities of state-of-the-art super-resolutionmicroscopy readout technologies (Sahl et al., 2017). For thispurpose, dip-pen nanolithography (Lee et al., 2002; Chai et al.,2011), electron beam lithography (Zhang et al., 2007), or STEDlithography (Harke et al., 2012; Wiesbauer et al., 2013; Fischeret al., 2015) have the advantage of a high resolution and fullfreedom of choice regarding the created protein patterns, but aretypically slow and require complex procedures and equipment.On the other hand, µCP has seen widespread adoption due toits simplicity and good performance. However, printing withPDMS stamps typically results in sagging of the interspacesor pairing for features significantly below 1µm (Schmid andMichel, 2000; Verschuuren, 2009; Kaufmann and Ravoo, 2010).Therefore, many creative methods were developed to circumventthe limitations of PDMS as a stamp material for nanocontactprinting.
One successful approach to create features below 100 nmmade use of silicone stamps with pyramidical features (Liet al., 2003; Filipponi et al., 2016). A drawback of this stamparchitecture is that only periodicities in the micrometer rangecan be realized. A different strategy to improve resolutionis to increase the Young’s modulus of the stamp material.This, however, can entail changes of the material propertiescompared to PDMS with respect to surface energy, as well
as the necessity for complex procedures in stamp production.Thus, the fundamental advantages of silicone such as ease-of-use,compliant contact, and low surface energy, are often abrogated.For instance, polyolefin plastomers foils can yield imprintsof superior quality in the sub-micrometer range compared toPDMS, however, the stamp development requires hot embossing(Schwaab et al., 2013). Therefore, global flatness is hard toachieve after demolding and requires equipment with a highcost of ownership especially for larger stamps (Shan et al., 2008).Furthermore, the material is not gas permeable which increasesthe appearance of trapped air bubbles, particularly in a manualprinting procedure. Another example is the use of a PDMSderivative with increased Young’s modulus of up to 9 MPa forprinting proteins with a periodicity down to 210 nm (Renaultet al., 2003). The density of proteins that could be achievedwith stamps featuring 100 nm pillars, however, was very low andimprints exhibited a number of defects.
Here, we present a nanocontact printing approach to fabricatehighly condensed 2D nanopatterns of proteins that has allthe advantages of silicone-based printing while avoiding itsdrawbacks. This is made possible by utilizing the novel stampmaterial X-PDMS in a 2-layer stamp architecture which has beendeveloped and to our knowledge so far only been applied forsubstrate conformal imprint lithography (SCIL) (Verschuurenet al., 2017). For SCIL, the authors developed a flexible siliconestamp consisting of a high modulus silicone (X-PDMS) onthe feature side and a soft low modulus silicone (PDMS) as abackplane. The different material properties of the two siliconesare a consequence of the polymer architecture: PDMS cures intoa two-dimensional network with a maximum Young’s modulusof 2.5 MPa, while the comparatively high stiffness of X-PDMSis achieved by the addition of tetrafunctional siloxanes, whichenable polymerization in three dimensions and thus increase theYoung’s modulus to up to 85 MPa (Verschuuren, 2009).
Thus, the 2-layer stamp combines the advantages of twoworlds: allowing features and a periodicity down to thenanometer range and having a long lifetime while still ensuringconformal contact between non-perfect surfaces over large areasand being easy to fabricate. We exemplify our approach bycreating a nanostructured antibody surface with feature sizesof 80 nm on a biocompatible background and evaluating thecreated protein patterns by atomic force microscopy (AFM) andstimulated emission depletion (STED) microscopy.
MATERIALS AND METHODS
Master DesignMasters for X-PDMS casting of stamps with 300 nm featuresize as well as pillars with 80 nm diameter were fabricated byphase transition mastering (PTM), a process developed andtypically applied for mastering of Blu-ray disks (Osato, 2003;Chang et al., 2011). PTM was performed with a PTR-3000master device modified for writing patterns different to Blu-ray designs. PTM allows relative freedom regarding the designof features, but has limits set, among others, by size of thelaser spot used for writing the patterns. For PTM, first, a layerof amorphous silicon (∼100 nm) was sputtered on an 8 inch
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Lindner et al. 2D Protein Nanopatterns
silicon wafer followed by a second layer of imperfect oxides oftungsten and molybdenum (phase transition material,∼100 nm)(Chang et al., 2011). Irradiation with a 405 nm laser changedthe state of the phase transition material from amorphous topolycrystalline, resulting in radial patterns produced by constantspinning of the wafer. The patterns were then developed usingtetramethylammonium hydroxide wet chemistry. Next, masterswere plated with nickel to ensure a smooth surface (Ra < 1 nm).During a resting period of 1 week, nickel was allowed to forman inert oxide layer to prevent abrasion during stamp casting.By carefully adjusting the sputtering parameters, the diameterof the wells in the master could be decreased down to 80 nmwidth (a design that will result in pillars of this diameter). Theavailable tools, however, did not allow to fabricate a master withpillar feature sizes smaller than 300 nm and a period significantlysmaller than 600 nm. The master for casting stamps featuring80 nm sized wells with a periodicity of 140 nm was thus boughtfrom Eulitha, Switzerland.
Stamp FabricationX-PDMS stamps were prepared following a protocol describedin Verschuuren (2009). Commercially available X-PDMS (SCILNanoimprint solutions, Philips, Netherlands) consists of twocomponents, A and B, which are of proprietary composition.The basis, however, has been published (Verschuuren, 2009):component A consists of a mixture of vinyl siloxanes including3D-branching Q-siloxanes, a platinum catalyst (platinum di-vinyl-tetra-methyl-di-siloxane), and a moderator (1,3,5,7-tetra-vinyl-1,3,5,7-tetra-methyl-cyclo-tetra-siloxane); component Bconsists of hybrid linear siloxanes, which are crosslinked uponmixing with component A. To achieve the highest Young’smodulus of ∼85 MPa (Verschuuren, 2009), components A andB were mixed in a ratio of 0.325:1 and subsequently degassedin a centrifuge with 2000 rpm for 3min. Three grams of themixture were poured on the wafer, spin coated at 2,000 rpmfor 30 s, and pre-cured for several minutes at 70◦C. Next, 3 gof Intermediate Layer (SCIL Nanoimprint solutions, Philips,Netherlands) was added, spin coated, and the stack was cured for10 days at 70◦C. Finally, a 2mm layer of PDMS was added andcured. The stamp surface was thoroughly washed with ethanol,iso-propanol, and deionized water (DIW) to remove residualmonomers. The quality of the stamps was evaluated by scanningelectron microscopy (SEM) in secondary electrons mode on aHitachi S-4000 with EMI compensation system MK3 (IntegratedDynamics Engineering, Germany). For SEM, small areas of thestamps were sputtered with a gold layer of∼20–30 nm.
Nanocontact PrintingGlass coverslips (#1.5, 24 × 60mm; Menzel R©, Fisher Scientific,USA) were coated withMix&GoTM Biosensor (Anteo DiagnosticsLtd, Australia) following the manufacturer’s protocol. Briefly, aglass coverslip was cleaned thoroughly in an ultrasonic bath withacetone and DIW. Directly afterwards, 100 µl of Mix&Go R© fluidwas pipetted onto the coverslip, incubated for 45min, washedwith DIW and dried in a stream of nitrogen.
Nanocontact printing was performed essentially followinga previously published protocol (Schütz et al., 2017). Briefly,
stamps were incubated with the desired protein (100µg/mlbovine serum albumin, BSA) or fibronectin (FNT, both fromSigma-Aldrich, USA) in phosphate-buffered saline (PBS, Sigma-Aldrich, USA) for 15min, rinsed thoroughly with distilled water,and dried in a N2 flow. After drying, the stamp was placed ona coated coverslip, pressed slightly to ensure good contact andincubated for 15min at room temperature. After removal of thestamp, patterns were measured immediately or stored at 4◦C inUniMailers R© Slide Mailers (VWR International GmbH, Austria)sealed with parafilm. Before measurements, the UniMailerswere warmed to room temperature before opening to avoidcondensation on the slides. Before and after each use, stampswere washed with ethanol, iso-propanol, and DIW to removedust particles and residual proteins.
AFM Characterization of NanopatternedSurfacesAtomic force microscopy (AFM) was performed on a DimensionEdge AFM (Bruker S.A.S., France) in tapping mode. OTESPA-R3(Bruker S.A.S., France) cantilevers with a spring constant of∼26N/m were used for imaging. AFM image analysis was performedin Gwyddion v2.49. Mainly, the correction algorithms “removepolynomial background,” “align rows using various methods,”and “correct horizontal scars (strokes)” were applied for leveling.Background subtraction was performed by taking the signal ofthe glass coverslip not covered by protein as a reference. Forquality assessment of protein transfer by printing, AFM imageswere converted to 16 Bit grayscale images and further analyzed inImageJ. For this, regular arrays were selected within the printedpatterns (Figure S1) corresponding to either regions with (“ON”)or without (“OFF”) stamp-surface contact. The mean gray valuesper pixel for each region, ION and IOFF, were used to calculate thecontrast C = ION−IOFF
ION. In AFM image analysis, care was taken to
perform the background subtraction the same way in all imagesto make contrast data comparable. Note though that contrastvalues are only intended as a means of relative comparisonbetween samples and are no absolute indicator of patternquality.
STED MicroscopyFor STED microscopy, goat anti-rabbit IgG antibody conjugatedwith STARRED (Abberior, Germany) was diluted 1:50 in PBS to afinal concentration of ∼20µg/ml. A Secure-Seal R© hybridizationchamber (Grace Biolabs, USA) was placed on top of the patternand filled with 50 µl of the antibody solution, incubated for15min and washed with 1ml of PBS to remove unboundantibody.
STED microscopy was performed on an inverted commercialSTEDmicroscope (Abberior Instruments, Germany) with pulsedexcitation and STED lasers. A 640 nm laser was used forexcitation and a 775 nm laser for stimulated emission. An oilimmersion objective with numerical aperture 1.4 (UPLSAPO100XO, Olympus, Japan) was used for imaging. The fluorescencesignal was collected in a confocal arrangement with a singlephoton counting avalanche photodiode using a 685/70 nmbandpass filter. The pulse repetition rate was 40 MHz andfluorescence detection was time-gated. The imaging parameters
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Lindner et al. 2D Protein Nanopatterns
used for acquiring STED images of W80 patterns were 40 µsdwell time, 41 µWexcitation laser power and 80 mW STED laserpower. The corresponding confocal images were recorded with50 µs dwell time and 2 µW excitation laser power. For bothSTED and confocal images, a pixel size of 10 nm, a pinhole of0.9 airy units and 3 line accumulations were used. The imagingparameters for acquisition of the STED images of W300 patternswere 40 µs dwell time, 84 µW excitation laser power and 94mW STED laser power. The corresponding confocal images wererecorded with 50 µs dwell time and 12 µW excitation laserpower. For both STED and confocal images, a pixel size of 20 nm,a pinhole of 1.0 airy units and 3 line accumulations were used.The power values refer to the power at the back aperture of theobjective lens.
RESULTS AND DISCUSSION
Our goal was to produce a patterned surface consisting ofnanometer-sized antibody dots and a biocompatible backgroundby nanocontact printing. Two different strategies can, inprinciple, be used to achieve such a surface architecture: (a)printing of an antibody (or a biofunctional protein such asstreptavidin) using stamps featuring a pillar profile followed bybackfilling with a protein for surface passivation, or (b) printingof a background protein using stamps featuring a well profile andbackfilling with antibody or streptavidin. In view of versatility ofthe produced patterns as well as long-term storability, we chose toadopt the latter approach to print BSA, a protein routinely usedfor surface passivation. The principle of surface preparation isillustrated in Figure 1.
Different strategies for surface preparation for proteinprinting have been proposed: plasma cleaning (Ricoult et al.,2014; MacNearney et al., 2016), epoxy functionalization (Sevcsiket al., 2015), streptavidin-coating (Lanzerstorfer et al., 2014) aswell as no further treatment of the glass substrate (Dirscherlet al., 2018). To achieve not only high imprint quality butalso good long-term storability of the printed nanopatterns,
we chose to functionalize the glass coverslips with Mix&Go R©
Biosensor, a polymer metal ion coating that does not reactwith water and allows strong attachment of proteins via aviditybinding. The first tested stamp layout featured wells of 80 nmdiameter and 140 nm periodicity (W80). Table 1 summarizesthe features of the different stamp layouts used in this study.A SEM image of the W80 stamp is shown in Figure 2A.AFM images of the BSA patterns printed with W80 revealeddefect-free imprints, with an average height of the printedprotein of ∼3 nm corresponding to a monolayer of BSA(Figures 2B,C).
For comparison, we printed patterns with a larger feature sizeof 300 nm and a periodicity of 600 nm (W300, Figures 2D–F).To quantitatively assess and compare imprint quality, thecontrast C between “ON” regions (where protein transfershould occur) and “OFF” regions (where no protein transfershould occur) was determined; contrast values are summarizedin Table 2. Printing of BSA using the W80 stamps wasreproducible with a mean contrast of 0.61, which was lowerthan for stamps with the W300 layout (C = 0.78). Thereare several possible explanations for this: first, out of the 10imprints analyzed for the W80 layout, 2 exhibited contrastvalues below 0.4. Such outliers were not observed for theW300 layout; they may be a consequence of the manualprinting process. Second, AFM imaging artifacts can compromisecontrast values. For example, edge effects caused by proteinsbeing dragged into the “OFF” areas are more pronounced
TABLE 1 | Features of the used stamps.
Stamp Type Diameter (nm) Period (nm) Depth (nm)
W80 Wells 80 140 100
W300 Wells 300 600 100
P80 Pillars 80 600 100
P300 Pillars 300 600 100
FIGURE 1 | Illustration of the nanocontact printing procedure. (A) Sketch of a stamp featuring a well (left) or pillar (right) layout. (B) The protein solution is incubated on
the X-PDMS/PDMS stamp. (C–E) After washing and drying, the stamp is brought into contact with a coverslip coated with MixandGo® Biosensor. The stamp is
pressed onto the coverslip, incubated and removed, leaving the protein pattern on the coverslip. (F) The quality of the imprint is determined by AFM imaging.
(G,H) After washing, the antibody is added to fill the interspaces, incubated and rinsed with water to remove unbound antibody.
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Lindner et al. 2D Protein Nanopatterns
FIGURE 2 | Images of the used stamps and printed BSA patterns. The first column shows SEM images of the different stamps used in this study. AFM images of
printed BSA are shown in the middle column; zoom-ins are shown in the insets. The white lines correspond to the line profiles shown in the third column. The rows
show data of the different stamp layouts as follows: (A–C) W80, (D–F) W300, (G–I) P80, (J–L) P300. Scale bar is 2µm in all images.
with decreasing feature size and lead to an overestimation ofthe height in “OFF” areas, resulting in an overall decreasedcontrast.
In contact printing, protein deposition does not depend on thecoherence of a light source or other long-range effects limiting thesize of the patterned area. The size limit for a patterned area isthus set, in principle, by the size of the master. While W80 andP80 masters were 30 × 30mm, W300 and P300 masters wereconsiderably larger (6 × 6 inches). Even with stamps producedfrom the latter, pattern quality was generally high over the wholeprinted area (Table 2). In a manual printing process, a practicallimitation is set by the dexterity of the experimenter, since thelikelihood of trapping air bubbles between stamp and substrateincreases with the stamp size.
The next step was to check whether reuse of the stamps woulddecrease the imprint quality. Even after 50 prints, however, thecontrast of the W80 imprints did not deteriorate (Figure S2A).Another important parameter is the storability of the printedpatterns. After 17 days of storage at 4◦C, we observed a smalldecrease in pattern contrast (Figure S2B).
For live cell applications of nanopatterned surfaces, it issometimes beneficial to promote cell adhesion in the areas nextto functionalized dots. In microcontact printing, fibronectinhas often been used for this task (Shen et al., 2008). Printingof fibronectin using the W80 stamp yielded patterns of poorcontrast (C = 0.21) and ring-like features (Figure S3A). Incomparison, the imprint qualities obtained with the W300stamp layout were of much better quality, similar to the onesobtained with BSA (Figure S3B). The poor performance offibronectin on the W80 stamps is most likely a consequenceof the size and properties of the fibronectin molecules: in acompact conformation, the molecular dimensions of fibronectinare ∼9 × 16 nm (Koteliansky et al., 1981), while the stretchedmolecule can reach lengths of up to 160 nm (Erikson et al., 1981).It is thus possible that fibronectin partially covers the 80 nm wellsthereby leading to a loss of resolution. Our results indicate thoughthat fibronectin can be used for printing structures with a welllayout of and above 300 nm feature size.
In some cases, it may not be feasible to print a backgroundprotein and fill with a functional protein. We thus tested the
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Lindner et al. 2D Protein Nanopatterns
TABLE 2 | Quality assessment of protein patterns.
Stamp layout Protein C n Comments
W80 BSA 0.61 ± 0.14 10 2 samples with C < 0.40
W300 BSA 0.78 ± 0.08 12
W 300 BSA 0.79 ± 0.02 3 3 different ROIs within one
6′′imprint
W80 BSA 0.58 ± 0.07 3 after 50 prints
W80 BSA 0.53 ± 0.03 3 after 17 days at 4◦C
W80 FNT 0.26 ± 0.11 9 0 samples with C > 0.50
W300 FNT 0.67 ± 0.05 14
P80 BSA 0.13 ± 0.28 6 1 sample with C > 0.75
P300 BSA 0.72 ± 0.02 7
P80 FNT 0.24 ± 0.34 9 2 samples with C > 0.75
P300 FNT 0.78 ± 0.02 6
W80 BSA/antibody 0.55 ± 0.04 3 C from STED images
W300 BSA/antibody 0.77 ± 0.03 3 C from STED images
n indicates the number of imprint replicates. Mean contrast values ± SD are given.
X-PDMS/PDMS stamp architecture for stamps featuring 80 nmpillars (P80, Figures 2G–I) and compared their performancewith 300 nm pillars (P300, Figures 2J–L). The performance of theP300 stamps was similar to the W300 ones, however, the meancontrast of the P80 patterns was markedly decreased compared toW80. Closer inspection of the data revealed that while 5 out of 6produced P80 patterns showed very poor contrast (C < 0.1), oneimprint was of high quality (C = 0.75; shown in Figures 2H,I).This heterogeneity in the performance of the P80 stamps mayoriginate from the manual printing process. Although AFMimages did not indicate permanent damage to the stamps afteruse, it is conceivable that excessive pressure during printingresults in a reversible collapse of the rather soft pillars leadingto a loss of contrast in the printed pattern. This may be avoidedby controlling the pressure during printing by using e.g., a SCILtool. Interestingly, printing of P80 fibronectin patterns yieldedsimilar results compared to BSA: 2 out of 9 imprints were of highquality (C > 0.75, Figure S4), while for the remainder printingwas not successful. This suggests that, with controlled pressure,robust printing of 80 nm features of fibronectin may be feasible.
We decided to continue our work using W80 BSA patterns.The next step in the process of surface preparation was backfillingwith a functional protein. The Mix&Go R© Biosensor we used toactivate the coverslips for protein attachment was specificallydesigned to preserve the functionality of antibodies (Ooi et al.,2014). Hence, fluorescently labeled antibody was directly addedto coverslips featuring W80 BSA patterns. Since the featuresizes of the nanopatterns are below the diffraction limit of light,conventional fluorescence microscopy cannot be used for qualityassessment. We thus employed STEDmicroscopy to visualize theproduced antibody nanopatterns. Both the W80 and the W300patterns are clearly visible in the STED images, while only theW300 features are discernible in the confocal images (Figure 3).Surfaces featuringW300 patterns appearmore homogeneous andwith less defects than surfaces with W80 patterns. Particularly, inthe W80 patterns, some dots seem to be devoid of or only weakly
FIGURE 3 | Evaluation of antibody nanopatterns with STED microscopy.
Representative areas of (A) W80 and (B) W300 BSA patterns backfilled with
Abberior STAR RED labeled antibody were imaged with STED microscopy.
The corresponding confocal images are shown in the bottom left corners. The
color map shows the number of detected photons. Scalebar is 1µm.
populated by fluorophores, while some dots seem excessivelybright. The contrast values determined for W80 and W300antibody patterns were 0.52 and 0.77, respectively.
Part of the difference between W80 and W300 patterns maybe due to a lower contrast already present in the W80 BSApatterns. Several factors can reduce the detected dot brightnessand thus the contrast: low degree of labeling of the antibodies,fluorophores lost due to bleaching by either the excitation laseror the high-intensity STED laser, fluorophores that are non-functional to begin with as well as a photon detection efficiencyof ∼70%. Considering the size of an antibody (10 × 15 nm),∼20–30 antibodies can maximally be accommodated in onewell with 80 nm diameter. Due to the stochastic distributionof antibodies on the surface, it is possible that the numberof fluorophores and thus detected photons differs significantlybetween individual dots produced with the W80 stamps. Withfeature sizes of 300 nm, this will lead to brightness heterogeneitieswithin individual antibody dots (as apparent from Figure 3B),whereas with 80 nm features, heterogeneities between dots willdominate.
CONCLUSION
We present here a nanocontact printing approach to generatenanopatterned protein surfaces that strikes a balance betweenresolution, simplicity, and speed, and validate our methodon the example of 80 nm sized antibody dots on a BSAbackground. Nanocontact printing with X-PDMS stamps on apolymer metal ion coated substrate neither requires a cleanroom facility nor cost-intensive equipment but allows thefabrication of reproducible highly condensed 2D protein patternson the nanoscale in a standard lab environment. For featuresizes of 80 nm, we found that stamps with a well layoutproduced high-contrast imprints with much higher fidelity thanpillar stamps. The quality of the printed protein patterns wasconsistent and robust over large areas, as assessed with AFMand STED microscopy. We showed that the stamps can bereused many times which further reduces the fabrication time of
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Lindner et al. 2D Protein Nanopatterns
nanopatterned surfaces. In addition, the signal-to-noise ratio ofthe produced patterns is high enough to support super-resolutionmicroscopy. While all experiments were performed manually,we believe that the contrast as well as the success rate of theimprints could be further improved with a printing tool similarto those used for SCIL. Even with standard lab equipment,the approach presented here is well-suited for a multitudeof applications in research laboratories, such as cell adhesionand protein interaction studies but may also prove particularlyuseful for printing large scale nanoarrays for biosensing or drugdiscovery. When combined with microfluidics for depositingdifferent capture proteins in parallel, the well layout presentedhere even allows the fabrication of multi-protein patterns on asingle biochip surface.
AUTHOR CONTRIBUTIONS
ML, IP, GS, and ES conceived and designed the experiments.ML, AT, and GF performed the experiments and analyzed the
data. WJ and JD designed, performed, and analyzed the STEDmicroscopy experiments. ML and AP designed and performedPTM mastering. ML and ES wrote the paper, while all otherauthors edited and revised the manuscript.
ACKNOWLEDGMENTS
We wish to thank Marc Verschuuren (Philips, Netherlands)for his help concerning the handling of X-PDMS and HarunSolak (Eulitha, Switzerland) for his support. We acknowledgefinancial support from the Austrian Science Fund (FWFprojects V538-B26) (ES, GF), P26337-B21 (ML, IP, GS),P25730-B21 (GS).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be foundonline at: https://www.frontiersin.org/articles/10.3389/fchem.2018.00655/full#supplementary-material
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Conflict of Interest Statement: The authors declare that the research was
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